Laboratory tests to diagnose Legionella pneumomonia are currently very insensitive, relying mainly on the culture of the organism, which may require up to 2 weeks, or on serologic conversion, which may take 2-4 weeks. In recent years, a urinary antigen assay has become commercially available, first as a radiometric assay but more recently in an enzyme immunoassay format. We have evaluated this assay using control organisms and random patient specimens. We found it to be highly specific, detecting only Legionella pneumophila serotype l. This assay is a welcome addition to diagnostic tests for Legionella, but it may be insufficient because of its high specificity, detecting only serotype 1. Recent published reports suggest that polymerase chain reaction (PCR) assays may provide a more sensitive technique for diagnosis. We have used a commercially available PCR assay for detection of Legionella from environmental samples as a starting point for developing a test for clinical specimens, particularly for sputum and bronchoalveolar lavage (BAL). The advantage of this procedure is its ability to detect almost all species of the genus Legionella, as well as to identify by specific probe the presence of L. pneumophila. Identification of other species would need further development Another significant advantage is the inclusion of a simple dot-blot assay, which we found sensitive enough to detect less than 100 colony-forming units per ml (using spiked specimens), yet yielded no false-negative reactions. Inhibition of PCR by blood in the specimens was removed by washing pelleted specimens in distilled water. For this study we screened all induced sputa and BAL specimens submitted for routine culture to determine the incidence of specimens deemed positive by this assay. For all PCR-positive assays, we looked at patient history, urinary antigen assay, culture, and serologic conversion to assess the validity of the PCR results. Of 126 patient specimens screened by PcR, l induced sputum and 3 BALs were positive by PCR. All 4 cases were validated as true positives either by culture of the organism or by serologic conversion. The entire PCR with hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 1012 days, and serologic conversion, 2-4 weeks. These studies have provided validation for in-house use of this procedure as an assay for the diagnosis of Legionella pneumonia; the assay will be offered by the Microbiology Service as a new molecular diagnostic test. A manuscript describing our studies and findings has been submitted for publication.